Novel Tissue Inhibitor of Metalloproteinase, TIMP-4, in Human Breast Cancer Growth and Metastasis

Tissue inhibitors of matrix metalloproteinase TIMPs are multifunctional proteins with both matrix metalloproteinases MMPs inhibitory effects and growth regulatory activity. TIMP-4, a novel human tissue inhibitor of metalloproteinase, was identified and characterized. Enzymatic kinetic studies revealed IC50 values of 19, 3, 45, 8, and 83 nM for MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, respectively. TIMP-4 has a strong inhibitory effect on the invasion of human breast cancer cells across reconstituted basement membranes. To determine if TIMP-4 can modulate the in vivo growth of human breast cancers, we administrated TIMP-4 both locally by transfection of the gene into breast cancer cells and systemically by a gene therapy approach with intramuscular injection of TIMP-4 expression plasmid. Overexpression of TIMP-4 locally in breast cancer cells inhibited the invasion of the cells in the in vitro and tumor growth in nude mice. However, here we report that systemic administration of human TIMP-4 by electroporation-mediated intramuscular injection of naked TIMP-4 DNA stimulates tumorigenesis of human breast cancer cells in nude mice. In consistence with tumor stimulatory effect, TIMP-4 upregulates Bcl-2 and Bcl-XL protein. TIMP-4 also protects apoptosis in human breast cancer cells in vitro and mammary tumors in vivo. A synthetic MMP inhibitor BB-94 did not have such anti-apoptotic effect. Analyses of TIMP-4 expression in human mammary specimens indicate that TIMP-4 protein is increased in mammary carcinoma cells compared with normal mammary epithelial cells. These data indicate an anti-apoptotic activity in breast cancer cells and a tumor-stimulating effect of TIMP-4 when administrated systemically.